The present invention relates in general to methods for controlling plasmid copy number and to plasmids constructed according to such methods. In particular, the present invention relates to methods and centromere-containing plasmids for the production of human erythropoeitin in yeast.
Microorganisms, including bacteria and yeasts, have been transformed by exposure to foreign genetic material in order to cause them to express exogenous genes (i.e. genes which do not naturally occur in the organism transformed). There is particular interest in the expression of mammalian gene products in yeast cells. Unlike bacteria, yeast cells share with mammalian cells the ability to glycosylate (i.e. to attach carbohydrates to) proteinaceous gene products. Furthermore, an extensive technology for the fermentation of yeast has been developed in the baking and brewing industries and yeasts appear on the list of organisms generally recognized as safe.
An exogenous gene may be prepared for introduction into a yeast cell by incorporating the gene into a vector. One type of vector is a circular, double-stranded DNA structure, called a plasmid, which replicates independently of the chromosomal DNA that forms the bulk of the hereditary material in the cell. Plasmids may be introduced into yeast cells by a process known as transformation.
A yeast-bacterial shuttle vector is a type of plasmid which contains a DNA replication initiation site, such as the chromosomal arsl sequence or the so-called 2.mu. origin of replication, which is recognized by the replication enzymes and factors of yeast cells. Yeast-bacterial shuttle vectors also contain an origin of replication from a bacterial plasmid which includes including an initiation site recognized by the replication enzymes and factors of bacterial cells. Beggs, Nature, 275, 104-109 (1979); Stinchcomb, et al., Proc. Natl. Acad. Sci. (USA), 77, 4559-4563 (1980).
Yeast-bacterial shuttle vectors are able to replicate in and may be selected in and recovered from bacteria, such as Escherichia coli (E. coli). It is generally convenient to construct and to amplify (i.e. multiply) plasmids in bacteria.
Yeast-bacterial shuttle vectors are also able to replicate in and may be selected in and recovered from yeasts, such as Saccharomyces cerevisiae (S. cerevisiae). In yeasts these plasmids may be used as transformation vectors for obtaining the expression of foreign DNA.
Yeast-bacterial shuttle vectors may contain a marker which permits selection of cells transformed by the vector. For example, a shuttle vector designated YRP7 contains the trpl gene, which codes for an enzyme called N-(5'-phosphoribosyl) anthranilate isomerase that is necessary for the production of the essential amino acid tryptophan. The trpl gene may be used as a marker by culturing cells in a tryptophan-deficient medium wherein only those cells having the trpl gene survive. Struhl, et al., Proc. Natl. Acad. Sci. (USA), 76, 1035-1039 (1979).
Despite their utility as vehicles for the expression of exogenous genes, yeast-bacterial shuttle vectors are more unstable than are recombinant plasmids in bacteria. For example, autonomously replicating plasmids containing an ars replicator are extremely unstable during cell division and are quickly lost under nonselective culture conditions. Stinchcomb, et al., Nature, 282, 39-43 (1979). One significant aspect of plasmid instability is that cultures of yeast may not be grown for many generations in a fermenter without extensive loss of the vector.
In bacteria, the maintenance of recombinant plasmids in host cells during host cell growth and the expression of heterologous genes incorporated in those plasmids may be facilitated by contol over two functions. Control over a partitioning function (i.e., the function which controls the distribution of plasmids among daughter cells during cell division) assures plasmid stability by preventing random loss of plasmids. Control over a replication function allows enhancement of heterologous gene expression by the amplification of plasmid copy number at a time close to cell harvest when the expression of toxic heterologous gene products does not interfere with host cell growth. Molin, et al., PCT Publication No. WO 84/01171.
Stable maintenance of plasmids in dividing cells requires that each daughter cell receive at least one copy of the plasmid. Thus, in the absence of a mechanism for accurately partitioning plasmids, plasmids are distributed on a purely random basis. One consequence of a random distribution of plasmids is that some daughter cells do not recieve a copy. Although a partition-defective plasmid may be maintained in a cell population by selecting for a marker (e.g., resistance to an antibiotic) associated with the plasmid, continuous application of selection pressure is difficult and expensive.
For example, selective markers useful in S. cerevisiae include a plasmid-borne, drug-resistant, type II dihydrofolate reductase gene (R-dhfr) [Miyajima, et al., Mol. Cell. Biol., 4, 407-414 (1984)] and the leu2-d, an allele of the Leu2 gene [Erhart, J. Bacteriol., 156, 625-635 (1983)]. Selection pressure on the R-dfhr markers is applied by growing cells in a medium including methotrexate. Plasmids having the leu2-d allele may be selected for by growth in leucine-deficient medium. Thus, cells containing at least one but an unknown number of marker plasmids may be selected. However, it is believed that for either of these markers, plasmid copy number is not known to provide a means for selecting for cells containing multiple copies of a plasmid and for selecting against cells containing a single copy of the plasmid.
In another approach to control of the partitioning function, incorporation of the par locus in a bacterial plasmid stabilizes an otherwise unstable plasmid by providing proper partitioning among daughter cells. Meacock, et al, Cell, 20, 529-542 (1980); Molin, et al., PCT Publication No. WO 84/01172.
Bacterial plasmid vectors are also available which provide for thermoinducible expression and temperature-regulated amplification of copy number. Uhlin, et al., U.K. Patent Application No. 1,557,774; Uhlin, et al., Gene, 6, 91-106 (1979); Remaut, et al., Gene, 22, 103-113 (1983). However, control of plasmid copy number, of the sort found in so-called "runaway" plasmids which amplify when released from some form of thermal inhibition on replication, is not available in yeasts. Dorfman, Genetic Engineering News, May/June, 8 (1983). Furthermore, even in bacteria, the large size of these runaway plasmids and their high basal copy number may divert the energy of the host cell from replication of DNA or mRNA to replication of the plasmid nucleic acid. A high basal copy number also increases the potential for poisoning of the host cell by toxic recombinant gene products. Thus, there is a need for some mechanism to balance the need for high copy number at expression and a low copy number during growth.
Centromere DNA sequences (CEN), isolated from chromosomes of S. cerevisiae, may be introduced into autonomously replicating yeast plasmids in order to stably maintain the plasmids through mitosis and meiosis. Bloom et al., Cell, 29, 305-317 (1982). Any one of five CENs, CEN3, CEN4, CEN5, CEN6 and CEN11, confers stability upon a plasmid into which it is introduced. Bloom, et al., J. Cell. Biol., 99, 1559-1568 (1984). CEN-containing plasmids are stably maintained through mitosis in the absence of selective pressure and segregate in the manner expected for centromere-linked genes. Plasmids containing a functional centromere are kept to a low copy number of 1 to 2 copies per cell. Clarke, et al., Nature, 287, 504-509 (1980). A block on replication at the CEN locus may prevent the completion of plasmid replication until anaphase. Tschumper et al., Gene, 23, 221-232 (1983). Such plasmids, containing a replication site and a centromere, are also called minichromosomes. Carbon, et al., U.S. Pat. No. 4,464,472.
The copy number control system of the endogenous 2.mu. plasmid of yeast provides for multiple rounds of plasmid replication when plasmid copy number is low. Plasmid copy number is thereby maintained at about 50 to 100 copies per cell. Beggs, Nature, 275, 104-109 (1978). Thus, although the 2.mu. system (which includes a replication sequence) prevents random loss of plasmids, it does not control plasmid segregation. Tschumper, et al., supra.
In plasmids containing both the CEN and 2.mu. copy number control mechanisms, the CEN mechanism dominates to result in normally segregating, composite plasmids of a copy number of 1 to 2 per cell. Tschumper, et al., supra. A CEN sequence may be inserted into a plasmid under the replication control of an ars in order to stabilize a CEN-containing plasmid. Srienc, et al., Mol. Cell. Biol , 5, 1676-1684 (1985).
In a different type of approach to coupling gene amplification with plasmid stability, a foreign gene is inserted into the downstream flanking region of the yeast copper chelatin gene on a minichromosome (i.e., a plasmid containing a yeast a CEN sequence) including an ars sequence. The locus containing the copper chelatin gene, the CUP1 locus, controls copper resistance in S. cerevisiae. The copper chelatin gene is present at a copy number which varies from one per cell in copper-sensitive strains, to as many as 10 or more tandemly iterated copies per cell in copper-resistant strains. Karin, et al., Proc. Natl. Acad. Sci. (USA), 81, 337-341 (1984). In the absence of copper, the copy number of a copper chelatin gene remains at an initial level, but when stressed by the introduction of copper, the copper chelatin gene and any exogenous gene inserted into its downstream flanking sequence form tandem multiple repeats in a minichromosome into which they are introduced. Fogel, et al., European Patent Application No. 96,491. However, this approach does not provide means for permitting the amplification of plasmid copy number.